Variations in DNA sequences have been associated with specific phenotypes related to athletic performance (33,43) and predisposition to injuries (20). Several studies have been conducted in the field of genetics of soccer, looking at the association between candidate gene variants (polymorphisms) and soccer-related phenotypes. However, often these studies yield contradictory results (14,17,22,25,38).
Various studies have found an association between genetic markers and specific phenotypes related to soccer performance. Particularly, studies have shown an association between genetic polymorphisms and soccer players' vertical jumps (22,27,30,32), sprint, (32), and endurance performances (32), as well as bone mass (7) and eccentric damage and catabolic state posteccentric training (31). An association between genetic variants and the status of being an elite soccer player, and/or elite endurance and sprint/power athletes (8,10,14,35), and with the predisposition to injuries in soccer (12,21,23,25,36) have also been shown.
A common A1470T (Glu490Asp) polymorphism (rs1049434) leading to the replacement of glutamic acid with aspartic acid has been identified in the MCT1 gene (also known as SLC16A1; location: 1p12) (28). The MCT1 T allele has been associated with reduction of lactate transport rate in red blood cells (28). When exercise intensity exceeds the lactate threshold, lactate production is greater than its clearance, leading to lactate accumulation in the muscle cell. Krustrup et al. (19) have analyzed muscle lactate during a soccer match and showed that the values were 15.9 ± 1.9 and 16.9 ± 2.3 mmol·kg−1 d.w. during the first and second halves, respectively. This, in turn, demonstrates the overall demanding nature of the game and the frequent use of glucose metabolism during a soccer match, untimely resulting in lactate accumulation. Transport of lactate across the plasma membrane of all cells is catalyzed by proton-linked monocarboxylate transporters (MCTs) (3,13,34).
Cupeiro et al. (4) have shown a higher capillary lactate accumulation during high-intensity circuit training in carriers of the T allele compared with their A-allele counterparts. More recently, the same group (5) has published an additional article in which they found the opposite, a higher lactate accumulation in AA participants.
In support to the later study, Fedotovskaya et al. (11) found that the frequencies of the A allele and AA genotype were significantly higher in endurance-oriented rowers compared with a control group, and Sawczuk et al. (39) found that the MCT1 TT genotype was associated with elite sprint/power athletic status.
More recently, Ben-Zaken et al. (2) found that the MCT1 T allele was more frequent among swimmers compared with runners, whereas we found that the MCT1 rs1049434 AA genotype is associated with higher incidence of muscle injuries in elite football players (unpublished data).
A possible explanation to the association between the MCT1 genotype and athletic performance might be related to the increased in lactate accumulation within the blood leading to induced expression of genes associated with muscle hypertrophy (39). Maximum gains in muscle hypertrophy are achieved by training regimens that produce significant metabolic stress (40). Buildup of metabolites, such as lactate, has been shown to significantly impact the anabolic processes (18).
The aim of this study was to examine the association between the MCT1 A1470T polymorphism and fat-free mass in young Italian soccer players, using plicometry to estimate each participant's fat-free mass percentage. Because the T allele was previously associated with sprint/power performance, and potentially growth in muscle mass, we have hypothesized that it would also be associated with an increase of fat-free mass in the soccer players.
Experimental Approach to the Problem
We used a cross-sectional design to examine the differences between the MCT1 A1470T polymorphism and fat-free mass percentage (%FFM) in 128 young elite Italian soccer players. The experimental period was March to April 2014. Each soccer player was subjected to anthropometric measurement. For each assessment, we followed the rules and techniques of measuring recommended by the International Working Group of Kinanthropometry, which were outlined by Ross and Marfell-Jones (37) and were adopted by the International Society for the Advancement of Kinanthropometry (ISAK).
Genomic DNA was extracted from saliva using a buccal swab and a QIAamp DNA Minikit (Qiagen, Hilden, Germany). The MCT1 A1470T polymorphism was analyzed by polymerase chain reaction following a previously published protocol (22). The primers used for amplification were as follows (29): sense primer 5′-ACACATACTGGGCATGTGGC-3′ (1455–1474); antisense primer 5′-AAA TCCCATCAA TGA ACAACTGGTATGATTTCCAC-3′ (1807–1841). Using the primers and the polymorphism (rs1049434) cited in the article by Cupeiro et al. (4), we searched the sequence on Genebank. Using the NebCuttertool (http://tools.neb.com/NEBcutter2/index.php), we individuated the restriction enzyme BccI, which allows for the distinction between the presence and absence of the A1470T polymorphism. This enzyme recognizes the sequence 3′-GGTAG-5′ and produces 3 fragments in the mutate sequence (TT: 14, 171, and 202 bp), whereas only 2 fragments (AA: 14 and 373 bp) were generated in the wild sequence. Heterozygote AT was individuated by 4 fragments: 14, 171, 202, and 373 bp (Figure 1).
The following variables were assessed for each athlete: height, weight, triceps, and subscapular skinfold (SF) thickness. The SF thicknesses were measured using the Lange caliper. A portable stadiometer (freestanding Magnimeter; Raven Equipment Ltd., Donmow, Essex, United Kingdom) was used to measure height. All measurements were performed twice, and the results were averaged. Technical errors of measurement met the required target levels for within and between measures (≤5% for SFs and ≤1% for height) (1). All participants were measured by the same trained anthropologist.
To estimate the %FFM, we have calculated, for each athlete, the percent body fat (%BF) first, using the Slaughter formula (41). The Slaughter equation has been developed and validated specifically for boys (children and adolescent) aged up to 17 years and predicts the %BF accurately with an overall high accuracy, minimal bias, and good precision with Dual-energy X-ray absorptiometry (DEXA) (24). It is expressed as follows:
If the sum of triceps and subscapular SF is >35 mm, the equation is as follows:
The weight expressed in kilograms of body fat was obtained using the following:
The weight expressed in kilograms of fat-free mass was obtained by subtracting body fat (kilogram) of the total body weight (kilogram).
Finally, %FFM was calculated using the following:
Pearson's χ2 and Fisher's exact tests were used to confirm that the observed genotype frequencies met the Hardy-Weinberg equilibrium distribution.
Differences between fat-free mass and MCT1 A1470T genotypes were tested using analysis of variance (using the Bonferroni post hoc test), and between the AA and T-dominant model (TT and AT genotypes) groups using unpaired t-test.
Linear regression analysis was performed to estimate the degree of variance in %FFM associated with the MCT1 A1470T genotypes. Statistica software (version 7.0) for Windows (Statsoft, Inc. 1984–2004, Tulsa, OK, USA) was used to perform all statistical evaluations. Significance was accepted when p ≤ 0.05.
One hundred twenty-eight young elite Italian soccer players (age: 16.3 ± 1.3 years; age range: 14.3–21.1 years; height: 173.1 ± 7.5 cm; weight: 64.5 ± 9.8 kg) were enrolled to the study; all were male and whites for ≥3 generations. All participants provided parental/guardian informed written consent, and the study protocol was approved by the Ethics Committee of the club and was in accordance with the Declaration of Helsinki for Human Research of 1974 (last modified in 2000). Athletes (soccer players) are members of the same team that competed at the National Level of the Official Italian Soccer Championship. Each participant completed a questionnaire concerning their training status and their parental origin. For all participants, the data were collected in the season training phase.
The MCT1 genotypes were in agreement with Hardy-Weinberg equilibrium distribution (p = 0.191). The genotype frequency of the MCT1 A1470T polymorphism is shown in Figure 2.
The AT genotype and the A allele were more frequent (AT = 56.3%; A = 61%) than the TT genotype and the T allele (TT = 10.9%; T = 39%), similar to previous reports in white populations (2,5,42). Participants' characteristics did not differ between genotypes with the exclusion of the participant's age, subscapular SF, and %FFM (Table 1). The soccer players with either the T allele or the TT genotype (the T-dominant model group) had a significantly lower subscapular SF value (p = 0.006) and were significantly younger than the carriers of the AA genotype (p = 0.018).
Soccer players with the TT genotype had a significantly higher %FFM than those with the AA genotype (p = 0.036).
The T-dominant model (TT and AT genotypes) showed significantly higher %FFM than the AA genotype (p < 0.001). The MCT1 A1470T genotype accounted for 5.4% of the variability in %FFM (R2 = 0.0548; F = 3.84; df = 1; p < 0.001).
The main finding in this study is that soccer players with the MCT1 TT genotype and the T allele had a significantly higher %FFM than their AA counterparts. Contrary to our expectations, the soccer players with the highest %FFM (T-dominant model group) were also the youngest. Interestingly, using regression analysis, we have also shown that the MCT1 A1470T polymorphism accounts for approximately 5.4% of the variance in %FFM among Italian soccer players. Hence, the MCT1 T allele may influence fat-free mass in young top-level soccer players. This is an interesting finding, particularly since fat-free mass is a key factor in determining the explosive power and its longitudinal development in pubertal soccer players (6).
During high-intensity muscle activity, such as repeated sprint, type 2, fast-twitch skeletal muscle fibers export accumulating lactate to adjacent type 1, slow-twitch muscle fibers to prevent acidosis, and to convert lactate to pyruvate. The transport capacity for lactate differs between individuals. MCT1 is required for lactate, produced by fast-twitch skeletal muscle fibres, to enter the myocytes for oxidation in heart and slow-twitch skeletal muscle fibers that uses lactate as a major respiratory fuel (15). Previously, individuals with the T allele had 60–65% less lactate transport rates than the mean normal (28). The MCT1 T allele was also related to diminished lactate transport by MCT1 (5), but its implication for athletic performance has not fully recognized. Recently, Sawczuk et al. (39) have suggested an explanation for the mechanism behind the MCT1-athletic performance association. The authors suggested that the association between MCT1 and athletic performance may be directly related to the increased accumulation of lactate within the blood that may induce the expression of genes associated with muscle hypertrophy, because increased lactate levels have been found to be associated with endogenous anabolic factors and muscle hypertrophy (3). This is, however, only a speculated explanation that needs to be further explored.
Here, we add to the existing literature that the T allele not only is associated with sprint/power performance, as shown by previous studies (11,39) but also to higher FFM% and there may be a link between this variant, hypertrophy, and fat mass.
An additional possible biochemical explanation to the genotype:phenotype association is that soccer players with the MCT1 T allele could have reduced their lactate transport into the less active muscle cells for oxidation, thus increasing blood lactate concentration. This condition could have induced the expression of muscle hypertrophy–associated genes (i.e., Insulin-like growth factor 1, growth hormone, etc.) (39) and acted as the primary contributor of osmotic changes in skeletal muscle, increasing cellular hydration and, as a consequence, augmenting the hypertrophic response during resistance training that relies heavily on anaerobic glycolysis (40).
Despite advances in our understanding of the genetic basis of athletic performance, there are limitations that have hampered the progression of genetic-based athletic research that need to be addressed (9,43). The primary limiting factor in genetic association studies is the need to recruit large groups of elite athletes to overcome the obvious barrier of large sample size for detecting genetic associations. To address this, DNA samples as well as muscle/blood/performance phenotypes from larger cohorts of soccer players and multicenter collaborations are required. We also suggest to specifically looking at the MCT1 A1470T and its relation with lactate accumulation/lactate transport and body mass phenotype to support our findings.
In conclusion, we found an association between the MCT1 T allele and %FFM in soccer player, and that this variant can explain ∼5% of the phenotype.
Soccer players with the MCT1 T allele (TT + AT genotypes) had significantly higher fat-free mass than soccer players with the AA genotype. MCT1 A1470T polymorphism should be considered as one of the polymorphisms that may influence soccer-related phenotypes. Discovering the complex relationship between gene variants and soccer performance may assist coaches to optimize training.
The authors thank all the staff and soccer players of the Cagliari Calcio Spa who participated in the study and who assisted with data collection. M. Massidda and C. M. Calò designed the research; M. Massidda, V. Bachis, L. Corrias, C. Culigioni, P. Cugia, M. Scorcu, and C. M. Calò performed the research; M. Massidda analyzed the data and wrote the article; C. M. Calò and N. Eynon participated in writing and reviewing the article.
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